May 29, 2016
- Hebert F.O.1,
- Grambauer S.1,
- Barber I.1,
- Landry C.R.1,
- Aubin-Horth N.1
- 1GigaScience
- GigaScience Press
External link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891850/
Protocol Citation: Hebert F.O., Grambauer S., Barber I., Landry C.R., Aubin-Horth N. 2016. RNA extraction protocol (Trizol). protocols.io https://dx.doi.org/10.17504/protocols.io.ew7bfhn
Manuscript citation:
Hébert FO, Grambauer S, Barber I, Landry CR, Aubin-Horth N, Transcriptome sequences spanning key developmental states as a resource for the study of the cestode Schistocephalus solidus, a threespine stickleback parasite. GigaScience, 2016, doi: https://doi.org/10.1186/s13742-016-0128-3
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: April 28, 2016
Last Modified: March 21, 2018
Protocol Integer ID: 2751
Keywords: total rna from flatworm, rna extraction protocol, flatworm, rna extraction, cestode schistocephalus solidus, total rna, threespine stickleback parasite, rna, reference transcriptome sequence resource, reference transcriptome sequence resource for the study, extraction, trizol
Abstract
This protocol describes how to extract total RNA from flatworms. It is from:
Hebert, F, O; Grambauer, S; Barber, I; Landry, C, R; Aubin-Horth, N (2016): Reference transcriptome sequence resource for the study of the Cestode Schistocephalus solidus, a threespine stickleback parasite. GigaScience Database. http://dx.doi.org/10.5524/100197
Troubleshooting
Safety warnings
Make sure to work under the fume hood for the Phenol and Chloroform extraction sections.
Before start
Pre-heat incubator at 65°C, clean bench surface and all the lab tools that will be used in the protocol (pipettes, tube holders, dissection forceps) with RNAse Zap/Rnase Wipes. Make sure that isopropanol + ethanol are at -20°C.
Phenol extraction
Add 1000 µL of Trizol per 50-100 mg of parsite tissues in a 2 mL eppendorf tube (RNAse free) and place the parasite tissues into their respective, labelled tube
Note
If a worm weights more than 100 mg, divide the whole worm into fractions of ~50-100 mg
Add 1 metal bead into each tube and place the tubes into a TissueLyzer. Run it at 30 Hz for 3 minutes
00:03:00
Centrifuge at 12,000 x g for 10 minutes at 4°C
00:10:00
Discard top layer of liquid formed in the eppendorf after the centrifugation (contains fatty acids)
Transfer supernatant into a new and labelled eppendorf tube (2 mL).
Incubate at room temperature for 5 minutes.
00:05:00
Chloroform extraction
Add 200 µL of chloroform per tube
Mix vigorously by inverting the tubes up and down for 1 minute. DO NOT VORTEX!
00:01:00
Centrifuge at 12,000 x g for 15 minutes at 4°C
00:15:00
Precipitation
Transfer with precaution the aqueous phase (supernatant) into a new 2 mL eppendorf tube
Note
Transfer small volumes (ex. ~7 x 100 µL)
Add 500 µL of RNAse free isopropanol (100%) and mix thoroughly
Incubate for 15 minutes at room temperature
00:15:00
Note
If yields are too low, incubate for 30-40 minutes
Centrifuge at 12,000 x g for 10 minutes at 4°C
00:10:00
Cleaning
Discard supernatant
Add 1000 µL of 75% ethanol (kept cold at -20°C) per tube
Vortex thoroughly and centrifuge at 7500g for 5 minutes at 4°C.
00:05:00
Discard supernatant and let the tubes dry out with the cap opened for 5-10 minutes
00:05:00
Resuspension
Add 20-50 µL of DEPC treated water into each tube
Place the tubes into the incubator (65°C) for 1 minute
00:01:00
Vortex thoroughly and repeat step 19 until RNA is completely dissolved.